Reduction and reoxidation of equine gonadotropin alpha-subunits.
Abstract: Ovine (o) and equine (e) LH alpha-subunits were reduced and reoxidized using conditions known to be effective for bovine and human alpha-subunits. The major product of oLH alpha refolding was alpha-subunit monomer. In contrast, eLH alpha formed a 121,000 mol wt aggregate. Monomeric eLH alpha was recovered, but in greatly reduced yield. To test the effects of carbohydrate variation on the aggregation of equine alpha-subunits, all of the equine gonadotropin alpha-subunits (eFSH alpha, eCG alpha, eLH alpha, and free alpha-subunit) were reduced and reoxidized. In each case, the major product was the 121,000 mol wt aggregate accompanied by monomeric equine alpha. Removal of carbohydrate by trifluoromethane sulfonic acid hydrolysis accentuated the tendency to aggregation during reoxidation. Most reduced-reoxidized deglycosylated eLH alpha did not enter a 12% sodium dodecyl sulfate-polyacrylamide gel. The highest LH receptor-binding activities were found in the alpha-subunit preparations, eLH alpha itself and pituitary free alpha-subunit. Operationally, the latter was separated from eLH in the last step of the eLH purification procedure; thus, LH contamination in this preparation is likely. Reduction and reoxidation reduced the LH receptor-binding activity of these two preparations to the level of LH activity observed in the eFSH alpha and eCG alpha preparations. We concluded that the majority of the LH receptor-binding activity observed in equine alpha-subunit preparations was due to contamination with eLH. We also obtained preliminary evidence that the amino-terminal and carboxy-terminal fragments of proteolytically "nicked" equine alpha-subunits refolded properly to form alpha monomer.
Publication Date: 1992-12-01 PubMed ID: 1280209DOI: 10.1210/endo.131.6.1280209Google Scholar: Lookup
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- Comparative Study
- Journal Article
Summary
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This research article explores the impact of the reduction and reoxidation process on equine gonadotropin alpha-subunits with a highlight on the encountered aggregations and the effects on LH receptor-binding activities.
Understanding the Experiment
- The study was conducted on ovine (o) and equine (e) LH alpha-subunits which were reduced and reoxidized using conditions that have been confirmed to be effective for bovine and human alpha-subunits.
- Rather than creating a dominant alpha-subunit monomer like the ovine LH subunit, the equine LH alpha-subunit produced a molecular weight aggregate of 121,000. A monomeric eLH alpha was recovered, though in a significantly reduced yield.
Impact of Carbohydrate Variation on Aggregation
- The researchers also investigated the effects of carbohydrate variations on the aggregation of equine alpha-subunits.
- All types of equine gonadotropin alpha-subunits, including eFSH alpha, eCG alpha, eLH alpha, and free alpha-subunit, were reduced and reoxidized.
- The major product was consistently a 121,000 molecular weight aggregate along with monomeric equine alpha.
- Enhanced aggregation during reoxidation was noted when carbohydrates were removed using trifluoromethane sulfonic acid hydrolysis.
Assessing LH Receptor-Binding Activities
- The highest LH receptor-binding activities were found in the alpha-subunit preparations, specifically eLH alpha and pituitary free alpha-subunit.
- The reduction and reoxidation, however, reduced the LH receptor-binding activity of these two preparations to the level of activity seen in eFSH alpha and eCG alpha preparations.
Conclusions and Key Findings
- The majority of LH receptor-binding activity in equine alpha-subunit preparations resulted from eLH contamination.
- Interestingly, the researchers also obtained preliminary evidence that the amino-terminal and carboxy-terminal fragments of proteolytically “nicked” equine alpha-subunits properly refolded to form an alpha monomer.
Cite This Article
APA
Bousfield GR, Ward DN.
(1992).
Reduction and reoxidation of equine gonadotropin alpha-subunits.
Endocrinology, 131(6), 2986-2998.
https://doi.org/10.1210/endo.131.6.1280209 Publication
Researcher Affiliations
- Department of Biological Sciences, Wichita State University, Kansas 67260-0026.
MeSH Terms
- Animals
- Electrophoresis, Polyacrylamide Gel
- Endopeptidases / metabolism
- Follicle Stimulating Hormone / chemistry
- Follicle Stimulating Hormone / metabolism
- Follicle Stimulating Hormone, beta Subunit
- Glycoprotein Hormones, alpha Subunit / chemistry
- Glycoprotein Hormones, alpha Subunit / metabolism
- Horses
- Macromolecular Substances
- Oxidation-Reduction
- Peptide Fragments / chemistry
- Peptide Fragments / metabolism
- Pituitary Gland / chemistry
- Protein Conformation
- Receptors, LH / metabolism
- Sheep
Citations
This article has been cited 1 times.- Butnev VY, Gotschall RR, Baker VL, Moore WT, Gout PW, Bousfield GR. Glycosylated equine prolactin and its carbohydrate moiety.. J Protein Chem 1996 Jul;15(5):413-26.
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